System, method and computer-accessible medium for providing wide-field superresolution microscopy

ABSTRACT

Exemplary embodiments of apparatus, system, method and computer-accessible medium using which at least one first pulsed electro-magnetic radiation is generated. Such radiation depletes at least one excited state of at least one molecule. Further, it is possible to generate at least one second electro-magnetic radiation based on the first pulsed electro-magnetic radiation(s). For example, the second electro-magnetic radiation(s) can have a pattern with a plurality of spots.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is based upon and claims the benefit of priority fromU.S. patent application Ser. No. 61/147,346, filed on Jan. 26, 2009, theentire disclosure of which is incorporated herein by reference.

FIELD OF THE DISCLOSURE

Exemplary embodiments of the present disclosure relate to system, methodand computer-accessible medium for providing information associated withsamples, and in particular to exemplary system, method andcomputer-accessible medium for utilizing wide-field superresolutionmicroscopy.

BACKGROUND INFORMATION

Recently, changes in optical microscopy have taken place, providing apossibility of sub-diffraction limited fluorescence imaging, also termed“nanoscopy.” (See Hell S. W., “Toward fluorescence nanoscopy”, Nat.Biotechnol. 2003, 21:1347-55). These advances can be referred toPALM/STORM (see Betzig E. et al., “Imaging intracellular fluorescentproteins at nanometer resolution”, Science 2006, 313:1642-5; and Rust M.J. et al., “Sub-diffraction-limit imaging by stochastic opticalreconstruction microscopy (STORM)”, Nat Methods, 2006, 3:793-5) and STED(see Westphal V., “Video-rate far-field optical nanoscopy dissectssynaptic vesicle movement”, Science 2008, 320:246-9; and Willig K. I. etal., “STED microscopy reveals that synaptotagmin remains clustered aftersynaptic vesicle exocytosis”, Nature 2006, 440:935-9).

The concept of PALM/STORM is to repeatedly photoactivate sparsefluorophores with a separation that is greater than the diffractionlimit and precisely resolve their locations using a Gaussian fittingprocedure. The STED concept operates by depleting the excitablefluorophores surrounding the center of the imaging spot using adonut-shaped beam. Both techniques have provided images of sub-cellulardetail with resolutions approaching 30 nm, heretofore only observable byelectron microscopy. (See Westphal V. et al., “Video-rate far-fieldoptical nanoscopy dissects synaptic vesicle movement”, Science 2008,320:246-9; and Huang B. et al., “Three-dimensional super-resolutionimaging by stochastic optical reconstruction microscopy”. Science 2008,319:810-3).

The above-described technologies have limitations. For example, thePALM/STORM procedure likely requires the excitation of rare events andcurrently takes many hours to achieve adequate signal to noise,prohibiting the imaging of living organisms. The STED procedures canwork faster (see Westphal V. et al, “Video-rate far-field opticalnanoscopy dissects synaptic vesicle movement”, Science 2008, 320:246-9),but may rely on the integrity of a donut beam to populate excitedstates. The lack of such integrity can limit or prevent sub-diffractionlimited imaging deep into tissues, as aberrations in tissue likelydestroy the shape of the donut beam. A technique capable of providingsub-diffraction limited imaging of intact or living tissues would likelyprovide a significant number of possibilities for nanoscopy in thebiological sciences. When applied to problems in human medicine, forexample, deep tissue nanoscopy in animal and human studies can providean improved understanding of the molecular mechanisms of tissue issues.

Accordingly, exemplary systems, methods and computer-accessible mediumproviding sub-diffraction limited imaging of intact or living tissuesmay be beneficial to overcome at least some of the above-describedissues and/or deficiencies.

SUMMARY OF EXEMPLARY EMBODIMENTS

Thus, at least some of the above-described issues and/or deficienciescan be addressed with the exemplary embodiments of the systems, methodsand computer-accessible medium according to the present disclosure.

Thus, exemplary embodiments of apparatus, system, method andcomputer-accessible medium can be provided which generate at least onefirst pulsed electro-magnetic radiation (e.g., using at least one firstarrangement). Such radiation depletes at least one excited state of atleast one molecule. Further, it is possible to generate at least onesecond electro-magnetic radiation (e.g., using at least one secondarrangement) based on the first pulsed electro-magnetic radiation(s).For example, the second electro-magnetic radiation(s) can have a patternwith a plurality of spots.

According to one exemplary embodiment, the pattern can be a specklepattern, and/or may be changeable. The first arrangement(s) can beconfigured to generate at least one third pulsed electro-magneticradiation which can excite the molecule(s) and precede the second pulsedelectro-magnetic radiation(s) in time. The molecule(s) can be providedin a biological structure. At least one third arrangement can also beprovided which is configured to forward the second and third radiationsto a substantially the same location on or in a biological structure.Further, at least one fourth arrangement can be provided which isconfigured to receive at least one fourth electro-magnetic radiationfrom the biological structure. Such exemplary fourth electro-magneticradiation(s) can have a wavelength which is different from a wavelengthof the second electro-magnetic radiation(s).

According to another exemplary embodiment of the present disclosure, thefourth arrangement(s) can generate an image of at least one portion ofthe biological structure based on the fourth electro-magneticradiation(s). The fourth arrangement(s) can include an array ofdetectors or at least one charged coupled detector. The fourtharrangement can generate a plurality of different images of a pluralityof portions of the biological structure having a plurality of distinctpatterns based on the fourth electro-magnetic radiation, and form afinal image from the plurality of the image.

According to a further exemplary embodiment of the present disclosure,the second arrangement can be controllable to change the pattern of thesecond electro-magnetic radiation(s). At least one fifth arrangement canbe provided which controls the second arrangement(s) to change thepattern of second electro-magnetic radiation(s). The fiftharrangement(s) can include (i) a spatial light modulator, (ii) a digitallight processor, (iii) a movable diffusing arrangement, and/or (iv) adigital mirror arrangement.

These and other objects, features and advantages of the exemplaryembodiment of the present disclosure will become apparent upon readingthe following detailed description of the exemplary embodiments of thepresent disclosure, when taken in conjunction with the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Further objects, features and advantages of the present disclosure willbecome apparent from the following detailed description taken inconjunction with the accompanying figures showing illustrativeembodiments of the present disclosure, in which:

FIG. 1 is a schematic diagram of an exemplary embodiment of a SpeckleSuperresolution Microscopy (“SSM”) apparatus according to the presentdisclosure;

FIG. 2A is a block diagram of an exemplary embodiment of asynchronization system according to the present disclosure whichsynchronizes two radiations by, e.g., sharing the same electro-magneticradiation source arrangement (e.g., a laser);

FIG. 2B is a block diagram of another exemplary embodiment of thesynchronization system according to the present disclosure whichsynchronizes two radiations by, e.g., utilizing an electronic controlthereof;

FIG. 3A is a block diagram of an exemplary embodiment of a patterngeneration system according to the present disclosure in a transmissionmode;

FIG. 3B is a block diagram of the exemplary embodiment of the patterngeneration system of FIG. 3A in a reflection mode;

FIG. 4A is an exemplary image of a simulation speckle pattern accordingto the present disclosure that illustrates available nulls in theexemplary pattern;

FIG. 4B is an exemplary simulation SSM image of a uniformly fluorescentbiological structure generated using the exemplary system according tothe present disclosure that indicates available excitation fluorophoresin the nulls in the pattern as shown in FIG. 4A;

FIG. 4C is an exemplary image of another simulation speckle patternaccording to the present disclosure that illustrates the available nullsin the exemplary pattern;

FIG. 4D is another exemplary corresponding simulation SSM image of auniformly fluorescent biological structure generated using the exemplarysystem according to the present disclosure that indicates the availableexcitation fluorophores in the nulls in the pattern as shown in FIG. 4C;

FIG. 5 is a schematic diagram of an exemplary embodiment of a detectionsystem according to the present disclosure that uses a magnificationsystem to image a sample on a wide-field detector; and

FIG. 6 is a flow diagram of an exemplary embodiment of an imageconstruction method according to the present disclosure for dataobtained from the SSM procedure and/or system.

Throughout the figures, the same reference numerals and characters,unless otherwise stated, are used to denote like features, elements,components or portions of the illustrated embodiments. Moreover, whilethe subject disclosure will now be described in detail with reference tothe figures, it is done so in connection with the illustrativeembodiments. It is intended that changes and modifications can be madeto the described exemplary embodiments without departing from the truescope and spirit of the subject disclosure as defined by the appendedclaims.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

According to the exemplary embodiment of the present disclosure whichcan be termed Speckle Superresolution Microscopy (“SSM”), the use oflaser speckle patterns to saturate excited states can be employed, whileleaving small focal locations at the nulls of the speckle patternavailable for excitation. One exemplary advantage of using speckle isthat, unlike STED, a coherent speckle pattern likely retains a strongzero-intensity null, even in the presence of a high degree of scatteringdeep into tissue. In addition, due to these strong nulls, the effectivesaturation factor can be about 2 orders of magnitude higher than that ofthe donut beam.

Unlike the PALM/STORM concept, the exemplary techniques and processesaccording to the present disclosure are likely not stochastic. Most ofthe fluorophores within the nulls may be available for excitation,beneficially resulting in a much higher signal-to-noise ratio (SNR).Moreover, according to the exemplary embodiment of the presentdisclosure, a bleaching stage-activation cycle may not be required. Forexample, with the exemplary SSM techniques according to the presentdisclosure, the nulls can be changed by altering the speckle pattern.

A schematic diagram of an exemplary embodiment of an SSM systemaccording to the present disclosure is shown in FIG. 1. Such exemplarysystem can include, e.g., two continuous wave or pulsed light sources110, 120, a speckle generator 122, a beam combinationelement/arrangement 125, a beam split element/arrangement 135, twolenses 191, 192 and a detector element/arrangement 150.

For example, using such exemplary system of FIG. 1, light 100 from thelight source 110 can be modulated by the speckle pattern generator 122to generate the light 101 with a speckle pattern. Two radiations 101,102 can be combined together by a beam combination element/arrangement125, and the combined radiations are illuminated on the substantiallysame position on or in the sample 180. The radiation 102 can excite atleast one molecule on or in the biological structure. In a pulse mode,the excitation pulsed radiation train 102 precedes the speckle pulsedradiation train 102 in time so that the speckle radiation may depletethe excited molecules of the biological structure on the focal plane. Insuch exemplary case, most of the fluorophores within the specklelocations on the pattern can be depleted to the ground states by theradiation 101. Only those fluorophores within the at least one nullbeyond the diffraction limit provided by the pattern may remainavailable for the fluorescence excitation. Thus, a fluorescent emissionsignal 103, which has the information of their locations on or in thebiological structure, can be provided through the lens 191 and used torecord, e.g., individually, with a super-resolution precision.

The fluorescent emission signal 103 can be transmitted back through thesame lens 191, and directed to a detection channel by the beam splitelement/arrangement 135. The lens 192 can magnify the image of thesample 180 to the detector element/arrangement 150.

According to an exemplary embodiment of the present disclosure, in orderto saturate the excited molecules, excitation and speckle radiations canbe synchronized. For example, two different synchronizationconfigurations can be obtained. FIG. 2A depicts a block diagram of anexemplary embodiment of an optical synchronization system, with tworadiations 201, 203 coming from the same electro-magnetic (e.g., initiallaser) source arrangement 210. The radiation from the laser source 210can be split into two parts by a beam splitter element/arrangement 211.One part can be the radiation (e.g., light) 201, which can be the lightfor a speckle generation. In another path, an electro-magnetic radiation(e.g., light) 202 can be transmitted through a wavelength converter 212to generate a radiation 203 have a particular excitation wavelength.

FIG. 2B shows another exemplary embodiment of the synchronization systemaccording to the present disclosure. For example, an electro-magneticradiation (e.g., light) source 251 can generate a radiation 261 for aspeckle generation, and another electro-magnetic radiation (e.g., light)source 252 can be used to generate an excitation radiation 262. Tworadiations 261, 262 can be synchronized by an electronic synchronizationarrangement 270.

To generate a changeable speckle pattern, e.g., two different exemplaryconfigurations can be implemented. For example, as shown in FIG. 3Awhich illustrates a block diagram of an exemplary embodiment of apattern generation system according to the present disclosure in atransmission mode, the incident light 301 is transmitted through achangeable speckle generator 330, and then, a speckle pattern 302 isgenerated. FIG. 3B shows a block diagram of another embodiment of thepattern generation system in which a changeable speckle generator 350can be used in a reflection mode to generate a speckle pattern 342 forthe incident light 341. One or both of the speckle generators 330, 350can be diffusing arrangement(s), a spatial light modulator, a digitallight processor, a digital mirror arrangement, or any other specklegenerator which can generate speckle patterns or alternatively anyarbitrary pattern. A plurality of distinct patterns can be produced byaltering the illumination's angle of incidence, and/or by rotating oneor moth of the generators 330, 350, changing the speckle pattern, oremploying any other means to generate different patterns. In suchexemplary manner, the locations of the nulls on the patterns can bechanged.

FIGS. 4A-4D show illustrations of two exemplary images associated withsimulation speckle a simulation patterns and two correspondingsimulation SSM images, respectively, that explain the exemplaryembodiment of the techniques according to the present disclosure. Forexample, a fully developed exemplary speckle pattern is shown in FIG.4A, demonstrating the exemplary characteristic graininess seen whenilluminating a scattering substance with a coherent laser. For example,the speckle pattern can illuminate a uniformly fluorescing sample suchthat most of the sample is driven into stimulated depletion of theground state, as shown FIG. 4B. In such exemplary case, most of thefluorophores within the locations represented by the nulls (black spots)likely remain available for fluorescence excitation. These dark spotscan be generally separated from each with a precision that is smallerthan the diffraction limit, and thus their locations may be individuallyresolvable with great precision using certain fitting procedures.

The locations of the dark spots can be changed to produce a new patternby altering the illumination's angle of incidence or creating a newspeckle pattern, e.g., as shown in FIGS. 4C and 4D. The exemplaryprocedure can be repeated by changing the pattern, and detecting anotherimage of a substantially different set of dark spots. The detection of aplurality of images and recombination of this plurality of images canprovide a resultant image of the structure. Wide field imaging andoptical sectioning can be accomplished by an exemplary method describedin, e.g., Ventalon C. and Mertz J., “Quasi-confocal fluorescencesectioning with dynamic speckle illumination”, Opt. Lett. 2005,30:3350-2, by measuring the variance or edges or high spatial frequencycontent of the speckle pattern as it is changed over time. Asupperresolution in the axial direction can also be facilitated by usingthe astigmatism STORM procedure discussed in, e.g., Huang B. et al.,“Three-dimensional super-resolution imaging by stochastic opticalreconstruction microscopy”. Science 2008, 319:810-3.

FIG. 5 shows a schematic diagram of an exemplary embodiment of adetection system according to the present disclosure that uses amagnification system to image a sample on a wide-field detector. Anexemplary fluorescent emission from at least one null beyond thediffraction limit of the speckle on or in the biological structure 550can be magnified by the lenses 501, 502 of the exemplary detectionsystem, and detected by a detection element/arrangement 555, such as acharged coupled detector (CCD) or an array of detectors. Themagnification can be designed or selected to have resolution thatexceeds the diffraction limit. Thus, the detection element/arrangement555 can record the image of the biological structure 550 with a pixelsize that can correspond to a portion of the image that is smaller thanthe exemplary diffraction limit.

FIG. 6 shows a flow diagram of an exemplary embodiment of an imagingconstruction procedure from data collection to completion of a fullimage acquisition according to the present disclosure. For example, aspeckle pattern can be generated (procedure 610), and an image can beacquired based on the speckle pattern (procedure 620). Then it can bedetermined if there are enough images (procedure 630). If not, then theprocessing returns to step 610. Otherwise, images are recombined togenerate a resultant image of structure. In this exemplary manner, animage of the biological structure can be obtained based on the specklepattern of the depletion radiation. Each speckle pattern can yield acertain number of image pixels based on the null locations of thespeckle pattern. Different speckle patterns can yield different imageswith only a few pixels illuminated therein. Therefore, by continuouslychanging the speckle patterns and acquiring multiple images, a resultantimage of the entire structure can be obtained by combining a pluralityof different images facilitated by a plurality of speckle patterns. Thiscan be done by the use of arithmetic combination(s), including addition,weighted summation, and/or the like.

The exemplary procedures to facilitate the operation of the exemplaryembodiments of the system according to the present disclosure and/orexecute the exemplary method shown in FIG. 6 can be performed bysoftware. Such software can be provided on or in a computer-accessiblemedium (e.g., hard drive, RAM, ROM, floppy disk, memory stick, SD car,mini-SD card, a plurality thereof and/or combination thereof). Thissoftware can be accessed, and then used by a processing arrangement(e.g., one or more computers) to program and/or configure suchcomputer(s) to execute the procedures defined and/or established by thesoftware on the processing arrangement to control the exemplarysystem/arrangement/method and obtain the results, as described hereinabove.

The peak power requirements and/or preferences for wide field SSM canlikely be greater than that of STED. To achieve significant STED action,the peak pulse energy density generally can be on the order of, e.g.,about 10-100 MW/cm2. Common pulsed lasers with sufficient peak power canbe used when the light is focused to a single spot with a high (e.g.,NA>1.0) lens. Utilizing the SSM systems, methods and/or procedures, anextended field can be illuminated, thus likely diminishing theirradiance by at least the square of the illuminated field diameter.Since the exemplary SSM systems, methods and procedures can process onephoton, the requirement/preference of power is likely lower than withthe multi-photon processes. Thus, it is possible to achieve thepreferable results using the exemplary SSM systems and/or procedureswith a conventional laser source. Moreover, it has been described thatSTED action can be attained at far lower powers by utilizing tripletstate relaxation (TREX). (See, e.g., Donnert G. et al.“Macromolecular-scale resolution in biological fluorescence microscopy”,Proc Natl Acad Sci US, 2006, 103:11440-5). An improved saturation factorfound with speckle illumination can assist with the exemplaryembodiments of the present disclosure, e.g., by diminishing the powerrequirements further. Nevertheless, if the exemplary wide field SSMprocedure is intractable due to the unavailability of appropriate lasersources, the exemplary SSM procedures and/or techniques can be conductedin a point scanning mode using conventional pulsed lasers and multipleor multi-node (i.e. quadrant) detectors.

According to the exemplary embodiments of the present disclosure, theexemplary SSM systems, methods and/or procedures are capable ofachieving a super-resolution image of a structure by speckle patternsaturation. For example, the exemplary embodiments of the presentdisclosure can provide exemplary system, method, computer-accessiblemedium and procedure for providing wide-field super resolutionmicroscopy in structures including scattering media using SSM.

The foregoing merely illustrates the principles of the invention.Various modifications and alterations to the described embodiments willbe apparent to those skilled in the art in view of the teachings herein.Indeed, the arrangements, systems and methods according to the exemplaryembodiments of the present disclosure can be used with and/or implementany OCT system, OFDI system, SD-OCT system or other imaging systems, andfor example with those described in International Patent ApplicationPCT/US2004/029148, filed Sep. 8, 2004 which published as InternationalPatent Publication No. WO 2005/047813 on May 26, 2005, U.S. patentapplication Ser. No. 11/266,779, filed Nov. 2, 2005 which published asU.S. Patent Publication No. 2006/0093276 on May 4, 2006, and U.S. patentapplication Ser. No. 10/501,276, filed Jul. 9, 2004 which published asU.S. Patent Publication No. 2005/0018201 on Jan. 27, 2005, and U.S.Patent Publication No. 2002/0122246, published on May 9, 2002, thedisclosures of which are incorporated by reference herein in theirentireties. It will thus be appreciated that those skilled in the artwill be able to devise numerous systems, arrangements and methods which,although not explicitly shown or described herein, embody the principlesof the invention and are thus within the spirit and scope of the presentdisclosure. In addition, to the extent that the prior art knowledge hasnot been explicitly incorporated by reference herein above, it isexplicitly being incorporated herein in its entirety. All publicationsreferenced herein above are incorporated herein by reference in theirentireties.

1. An apparatus comprising: at least one first arrangement configured togenerate at least one first pulsed electro-magnetic radiation whichexcites at least one molecule; and at least one second arrangement whichis configured to generate at least one second electro-magnetic radiationbased on the at least one first pulsed electro-magnetic radiation,wherein the at least one second electro-magnetic radiation has a specklepattern with a plurality of spots, and which depletes at least oneexcited state of the at least one molecule.
 2. The apparatus accordingto claim 1, wherein the pattern is changeable.
 3. The apparatusaccording to claim 2, further comprising at least further arrangementconfigured to (i) receive further radiation from a sample based on thefirst and second radiation and generate at least image associated with adepth of the sample, and (ii) measure at least one of a variance, edgesor a high spatial frequency content of the at least one image as it ischanged over time.
 4. The apparatus according to claim 1, wherein the atleast one molecule is provided in a biological structure.
 5. Theapparatus according to claim 1, further comprising at least one thirdarrangement which is configured to forward the further and secondradiations to a substantially the same location on or in a biologicalstructure.
 6. The apparatus according to claim 5, further comprising atleast one fourth arrangement which is configured to receive at least onethird electro-magnetic radiation from the biological structure, the atleast one third electro-magnetic radiation has a wavelength which isdifferent from a wavelength of the at least one first electro-magneticradiation.
 7. The apparatus according to claim 6, wherein the at leastone fourth arrangement generates an image of at least one portion of thebiological structure based on the at least one third electro-magneticradiation.
 8. The apparatus according to claim 7, wherein the at leastone fourth arrangement includes an array of detectors or at least onecharged coupled detector.
 9. The apparatus according to claim 6, whereinthe at least one fourth arrangement generates a plurality of differentimages of a plurality of portions of the biological structure having aplurality of distinct patterns based on the at least one thirdelectro-magnetic radiation, and forms a final images from the pluralityof the image.
 10. The apparatus according to claim 9, wherein the atleast one fourth arrangement is further configured to process andassociate at least one image of the images with one or morepredetermined functions.
 11. The apparatus according to claim 10,wherein the at least one fourth arrangement is further configured todetermine one or more center of the one or more predetermined functions.12. The apparatus according to claim 10, wherein the at least one imageis acquired associated with a plurality of the one or more of thecenters.
 13. The apparatus according to claim 10, wherein the at leastfourth arrangement processes a plurality of the images based on aplurality of the centers to form a final image.
 14. The apparatusaccording to claim 6, wherein the at least one third electro-magneticradiation is a florescence radiation.
 15. The apparatus according toclaim 1, wherein the at least one second arrangement is controllable tochange the pattern of the at least one second electro-magneticradiation.
 16. The apparatus according to claim 1, further comprising atleast one fifth arrangement which controls the at least one secondarrangement to change the pattern of the at least one secondelectro-magnetic radiation.
 17. The apparatus according to claim 16,wherein the at least one fifth arrangement includes at least one of (i)a spatial light modulator, (ii) a digital light processor, (iii) amovable diffusing arrangement, or (iv) a digital mirror arrangement. 18.A method comprising: using at least one hardware arrangement, generatingat least one first pulsed electro-magnetic radiation which excites atleast one molecule; and generating at least one second electro-magneticradiation based on the at least one first pulsed electro-magneticradiation, wherein the at least one second electro-magnetic radiationhas a speckle pattern with a plurality of spots, and which depletes atleast one excited state of the at least one molecule.
 19. Acomputer-accessible medium which has software thereon, wherein, when acomputing arrangement retrieves and executes the software, the computingarrangement is configured to perform procedures comprising: causing ageneration of at least one first pulsed electro-magnetic radiation whichexcites at least one molecule; and causing a generation of at least onesecond electro-magnetic radiation based on the at least one first pulsedelectro-magnetic radiation, wherein the at least one secondelectro-magnetic radiation has a speckle pattern with a plurality ofspots, and which depletes at least one excited state of the at least onemolecule.